Health assessment of Holothuria scabra (Jaeger, 1938) from pond culture in Thailand: Size distribution and gonadal development

Are the annexin V microbeads, a real solution to sperm DNA fragmentation?

2282 EFFECTS OF SMOKING, ALCOHOL CONSUMPTION AND AGE ON SEMEN QUALITY IN MEN EVALUATED FOR SUBFERTILITY

Micro-osteoperforations accelerate orthodontic tooth movement by stimulating periodontal ligament cell cycles.

2283 CONCORDANCE AMONG SPERM DNA INTEGRITY ASSAYS AND SEMEN PARAMETERS

Matrix metalloproteinases (MMPs) play intricate roles in cancer progression; some promote invasion and angiogenesis while others suppress tumor growth. For example, human MMP-26/endometase/matrilysin-2 was reported to be either protective or pro-tumorigenic. Our previous reports suggested pro-invasion and anti-inflammation properties in prostate cancer. Here, we provide evidence for a protective role of MMP-26 in the prostate. MMP-26 expression levels in androgen-repressed human prostate cancer (ARCaP) cells, transfected with sense or anti-sense MMP-26 cDNA, are directly correlated with those of the pro-apoptotic marker Bax. Immunohistochemical staining of prostate cancer tissue samples shows similar protein expression patterns, correlating the expression levels of MMP-26 and Bax in benign, neoplastic, and invasive prostate cancer tissues. The MMP-26 protein levels were upregulated in high grade prostate intraepithelial neoplasia (HGPIN) and decreased during the course of disease progression. Further analysis using an indirect terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that many tumor cells expressing MMP-26 were undergoing apoptosis. This study showed that the high level of MMP-26 expression is positively correlated with the presence of apoptotic cells. This pro-apoptotic role of MMP-26 in human prostate cancer cells and tissues may enhance our understanding of the paradoxical roles of MMP-26 in tumor invasion and progression.

Dear Editor: In recent years, laser toning using low fluence, multiple-passed, Q-switched, 1,064-nm neodymium:yttrium-aluminum-garnet (Nd:YAG) lasers has gained popularity in Asian countries for the treatment of melasma. However, laser toning often results in hyperpigmentation or mottled depigmentation1,2,3. Therefore, it is critical to determine the optimal conditions for laser toning, including pulse type and irradiation energy density. In our previous study using a zebrafish model4, we suggested the concept of subcellular selective photothermolysis (SSP) of melanosomes. The recently introduced dual-pulse, 1,064-nm, Q-switched Nd:YAG laser has received considerable attention. We performed a pilot study to investigate the influence of pulse type on SSP of melanosomes. For this purpose, we used two Q-switched Nd:YAG lasers with different pulse types: (1) conventional pulse (Spectra VRMIII; Lutronic Corp., Goyang, Korea) and (2) dual-pulse laser (Pastelle-PTP mode; Won Tech., Daejeon, Korea). The conventional pulse-type laser had a wavelength of 1,064 nm; fluence of 0.4, 0.5, and 1.0 J/cm2; and a spot size of 7 mm. The dual-pulse laser had a wavelength of 1,064 nm; fluence of 0.4, 0.5 and 1.0 J/cm2; and a spot size of 7 mm. We irradiated the caudal peduncle of adult zebrafish with a single light pulse. To identify the fluence level that sufficiently destroys melanosomes, zebrafish were irradiated at fluence levels of 0.5 and 1.0 J/cm2 with both pulse types, with one zebrafish subjected to each condition. We observed and analyzed temporal changes in melanosomes at the irradiation site, and we examined the tissues 1 week after irradiation when all of the pigment debris had disappeared. The images were developed into black and white photographs to accentuate pigment-free lesions. The relative pigmentation density after laser irradiation was quantified using Quantity One 1-D analysis software (version 4.6.3; Bio-Rad Inc., Hercules, CA, USA). According to the SSP concept, we stained the zebrafish skin tissue with 4',6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) 1 day after irradiation at fluence levels of 0.4 and 0.5 J/cm2 with both pulse types to test whether the laser pulse type has an effect on melanophores and their adjacent cells. The experimental protocols were approved by the Korea University Animal institutional review board (No. KUIACUC-2013-172). A relative pigmentation density less than 1.0 was observed 1 week after irradiation with light at fluence levels of 0.5 and 1.0 J/cm2 with both pulse types. Regeneration of melanosomes was observed at 4 and 8 weeks of irradiation at 0.5 J/cm2 (Fig. 1). A similar change was observed when irradiation was performed at 1.0 J/cm2 with both pulse types. DAPI and TUNEL staining results are shown in Fig. 2. No apoptotic cells were detected for either pulse type at a fluence level of 0.4 J/cm2. As demonstrated in our previous study, apoptotic cells appeared after irradiation with the conventional pulse type at a fluence of 0.5 J/cm2; however, no apoptosis was observed at 0.5 J/cm2 for the dual-pulse type laser. Fig. 1 Changes over time in melanosomes at the site of laser irradiation (×12.5). For the conventional and dual-pulse type lasers, a fluence of 0.5 J/cm2 was sufficient for the near complete elimination of adult zebrafish melanosomes. Melanosomes regenerated ... Fig. 2 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining of zebrafish skin tissue 1 day after laser irradiation (×20). DAPI staining (blue) shows normal and apoptotic nuclei. TUNEL ... These results indicate that a fluence of 0.5 J/cm2 with either pulse type is sufficient for the near complete elimination of adult zebrafish melanosomes. However, a distinct laser pulse type-dependent difference in necrosis and apoptosis of melanophores and other cells was observed at a fluence of 0.5 J/cm2. The exact reason underlying the results obtained with the dual-pulse laser remains unclear. The dual-pulse method cuts the pulse into two half-fluence pulses, and this produces a 140-µs interval between the dual-pulse beams. Our results suggest that the dual-pulse laser destroys melanosomes with a sub-threshold peak power. Therefore, the dual-pulse method could correspond with the SSP hypothesis; however, further studies are needed to confirm our results. Through SSP, the relatively low irradiation energy could limit thermal diffusion effects to melanosomes without affecting melanophores or other cells. It is presumed that apoptosis of melanocytes after laser irradiation at threshold and supra-threshold fluencies can lead to pigmentary complications1,2. We speculate that SSP at a low fluence level could lower the risk of side effects associated with laser toning. In summary, this study suggests that the laser pulse type influences SSP of melanosomes. Future laser toning studies should focus on establishing proper treatment methods that involve pulse type and irradiation energy, to support selective photothermolysis at a subcellular level with the goals of optimizing efficacy and minimizing complications.

목적: 본 연구는 망막색소상피층에 색소가 존재하는 mouse에서 청색광으로 인해 광수용세포 손상이 일어날 수 있는지 확인하고, 광수용세포 중 특이적 세포에서 세포사멸이 유도되는지 조사하여 청색광에 의해 야기될 수 있는 연령관련 황반변성의 기전 규명과 치료제 개발에 도움이 되고자 진행되었다. 방법: C57black mice를 24시간 암순응 시켜 463 nm의 청색광을 <TEX>$2800{\pm}10lux$</TEX>로 조사한 후 1일, 3일, 7일째에 안구를 적출하였다. 청색광의 자극은 GFAP(Glial fibrillary acidic protein)단백질의 발현을 이용하여 확인하였고, 광수용세포의 세포사멸은 TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling)을 사용하여 분석하였다. Western blotting으로 ERK(Extracellular signal-regulated kinases), c-JUN, SRC(Sarcoma) 단백질 발현을 확인하였고, 막대세포와 원뿔세포의 손상 정도를 비교하기 위해 면역염색으로 분석하였다. 결과: 청색광을 조사한 후 1, 3, 7일이 지난 망막은 대조군 보다 전체적으로 두께가 감소하였고, 각 얼기층보다 핵층에서 두께 감소를 확인할 수 있었다. 또한 청색광을 조사한 후 1일 지난 Muller glia에서 GFAP 단백질이 증가하는 것을 확인하였다. TUNEL 염색에서는 청색광을 조사한 후 1일 지난 망막의 광수용세포에서 가장 많은 발현을 보였다. 세포사멸 기전 과정 중 하나임을 확인하기 위해 ERK, c-JUN, SRC 단백질 활성을 확인한 결과 청색광을 조사한 망막에서 phosphorylated ERK는 증가하였고 phosphorylated SRC는 조사 후 1일에서만 증가를 나타내었으며, 반대로 phosphorylated c-JUN은 조사 후 1일에서만 감소하였다. 청색광을 조사한 망막에서 막대세포 발색단인 로돕신과 원뿔세포의 발색단인 옵신이 감소하였으며, 옵신의 감소량은 로돕신의 감소량보다 큰 것을 확인하였다. 결론: 본 연구는 청색광이 망막에 손상자극을 주고, ERK와 SRC 신호전달과 관련하여 광수용세포의 세포사멸을 일으킬 수 있으며 청색광이 광수용세포 중 원뿔세포의 세포사멸을 직접적으로 유도하여 망막 손상을 야기할 수 있다는 가능성을 제시하였다. Purpose: The study was conducted to determine that photoreceptors of mouse having pigment in RPE(retinal pigment epithelium) can be damaged by blue-light and apoptosis of specific cells among photoreceptors are induced by blue-light, and to assist the investigation of AMD(Age-related macular degeneration) mechanisms and development of AMD drugs. Methods: C57Black mice were injured by irradiating <TEX>$2800{\pm}10lux$</TEX> of 463 nm LED for 6 hours after 24 hours dark adaptation and eyes were enucleated 1, 3, 7 days. Damage of retina induced by blue-light was determined by western blotting GFAP(Glial fibrillary acidic protein) expression. In the light-injured retina, cell death of photoreceptors was determined by TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. ERK(Extracellular signal-regulated kinases), JNK, and SRC(sarcoma) expression were assessed by western blotting to determine regulated pathway. Blue light-injured retina were immunostained with antibodies against Opsin and Rhodopsin as markers of photoreceptors to compared the damage cone cells with rod cells. Results: After 1, 3 and 7 days from exposure to blue-light, thickness of retina was more decreased than control, and more decreased at nuclear layer than at outer plexiform layer and GFAP expression was increased day 1 after blue-light injured. While phosphorylated ERK and SRC protein expressions at day 1 were increased after blue-light injured, phosphorylated c-JUN was decreased. Fluorescence intensity analysis showed that markers of cone and rod cells were decreased after blue-light injured and Opsin was more decreased than Rhodopsin. Conclusions: The study suggests possibilities that the blue-light promotes retinal damage and causes apoptotic cell death via ERK and SRC pathway in mouse retina, and blue-light retinal damage is more induced cone cells apoptosis than rod cells directly.

DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction

Sperm chromatin damage has been associated with male infertility, increased risk for spontaneous abortion, and poor embryo development. Available methods for detecting chromatin damage render the sperm no longer suitable for clinical use. Early apoptotic events resulting in chromatin damage are associated with increased permeability of the cell membrane to large ions. We propose the use of a large fluorescent organic cation, proprietary fluorochrome (PF-1), for fluorescence-activated cell sorting (FACS) for negative selection of sperm without chromatin damage. Sperm with chromatin damage are PF-1 positive. Performance of cell sorting by PF-1 was verified with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) after FACS on PF-1(+) and PF-1(-) subpopulations. Whereas 19.5% of PF-1 positive sperm were TUNEL positive only 1.5% sperm in the PF-1(-) fraction were TUNEL positive (p < 0.00001). TUNEL values below 1.9% were considered background fluorescence. Post-sorting motility and vitality were 49.4% (SD: 12.5) and 65.0% (SD: 14.99), respectively. Proprietary fluorochrome activated sperm sorting may decrease or most likely eliminate all of TUNEL positive sperm without adverse effects on viability, providing a new therapeutic avenue for men with a high percentage of TUNEL positive sperm. Further research is needed to determine if the reduction in TUNEL positive sperm using PF-1 will improve in vitro fertilization (IVF) outcomes.

Oligoasthenoteratozoospermia (OAT) is the most prevalent male infertility condition that is mainly caused by sperm DNA fragmentation (SDF). This study compared the sensitivity and effectiveness of two different approaches for analyzing SDF in patients with OAT: sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In this study, which received ethical committee approval, participants were divided in to normal and OAT groups (n=20 for each). both TUNEL and SCD assays were used to analyze the sperm DNA fragmentation. And Malondialdehyde (MDA) levels was measured to determine levels of lipid peroxidation in the seminal plasma. The TUNEL assay showed better ability to predict OAT patients than that of the SCD. For our patient population, the projected cut-off points for the DNA fragmentation index of 29% and 19% were reported using the TUNEL and SCD tests, respectively. Seminal levels of MDA were significantly higher in the OAT group (P=0.002) than that of control group. OAT patients showed higher MDA levels of seminal plasma and DNA fragmentation than the control group. Although sperm DNA fragmentation can be detected with high efficiency and sensitivity using both TUNEL and SCD assays, the TUNEL test was found to be a more accurate predictor for OAT patients.

Aims/IntroductionCell death‐inducing DFF45‐like effector C (CIDEC) was proven to be closely associated with the development of insulin resistance and metabolic syndrome. We aimed to investigate whether CIDEC gene silencing could alleviate pulmonary vascular remodeling in a type 2 diabetes rat model.Materials and MethodsWe built a type 2 diabetes rat model. An adenovirus harboring CIDEC small interfering ribonucleic acid was then injected into the jugular vein to silence the CIDEC gene. After hematoxylin–eosin and Sirius red staining, we detected indexes of the pulmonary arterioles remodeling. Immunohistochemical staining of proliferating cell nuclear antigen was used to evaluate the pulmonary arterial smooth muscle cell proliferation. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling reaction and western blotting. The levels of signaling pathway proteins expression were measured by western blotting analyses.ResultsHistological analysis of the pulmonary artery showed that the thickness of the adventitia and medial layer increased notably in type 2 diabetes rats. Immunohistochemistry showed that more proliferating cell nuclear antigen‐positive pulmonary arterial smooth muscle cells could be seen in type 2 diabetes rats; and after CIDEC gene silencing, proliferating cell nuclear antigen positive cells decreased accordingly. Cleaved caspase‐3 and cleaved poly (adenosine diphosphate‐ribose) polymerase measured by western blotting showed increased apoptosis with overexpressed CIDEC in diabetes. Terminal deoxynucleotidyl transferase dUTP nick end labeling reaction showed that the apoptosis mainly occurred in endothelial cells. Western blotting analysis showed CIDEC overexpression in rats with diabetes, and phosphorylated adenosine 5′ monophosphate‐activated protein kinase‐α expression was significantly decreased. After CIDEC gene silencing, the expression of phosphorylated adenosine 5′ monophosphate‐activated protein kinase‐α was upregulated.ConclusionsThe CIDEC/5′ monophosphate‐activated protein kinase signaling pathway could be a potential therapeutic candidate against pulmonary vascular diseases in type 2 diabetes patients.

Abnormally enhanced cystic fibrosis transmembrane conductance regulator-mediated apoptosis in endometrial cells contributes to impaired embryo implantation in controlled ovarian hyperstimulation

Focal cortical infarction causes neuronal apoptosis in the ipsilateral nonischemic thalamus and hippocampus, which is potentially associated with poststroke cognitive deficits. TSPO (translocator protein) is critical in regulating mitochondrial apoptosis pathways. We examined the effects of the novel TSPO ligand 2-(2-chlorophenyl) quinazolin-4-yl dimethylcarbamate (2-Cl-MGV-1) on poststroke cognitive deficits, neuronal mitochondrial apoptosis, and secondary damage in the ipsilateral thalamus and hippocampus after cortical infarction. One hundred fourteen hypertensive rats underwent successful distal middle cerebral artery occlusion (n=76) or sham procedures (n=38). 2-Cl-MGV-1 or dimethyl sulfoxide as vehicle was administrated 2 hours after distal middle cerebral artery occlusion and then for 6 or 13 days (n=19 per group). Spatial learning and memory were tested using the Morris water maze. Secondary degeneration and mitochondrial apoptosis in the thalamus and hippocampus were assessed using Nissl staining, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling, JC-1 staining, and immunoblotting 7 and 14 days after surgery. Infarct volumes did not significantly differ between the vehicle and 2-Cl-MGV-1 groups. There were more neurons and fewer glia in the ipsilateral thalamus and hippocampus in the vehicle groups than in the sham-operated group 7 and 14 days post-distal middle cerebral artery occlusion. 2-Cl-MGV-1 significantly ameliorated spatial cognitive impairment and decreased neuronal death and glial activation when compared with vehicle treatment (P<0.05). The collapse of mitochondrial transmembrane potential and cytoplasmic release of apoptosis-inducing factors and cytochrome c was prevented within the thalamus. Caspase cleavage and the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling+ or Nissl atrophic cells were reduced within the thalamus and hippocampus. This was accompanied by upregulation of B-cell lymphoma 2 and downregulation of Bax (P<0.05). 2-Cl-MGV-1 reduces neuronal apoptosis via mitochondrial-dependent pathways and attenuates secondary damage in the nonischemic thalamus and hippocampus, potentially contributing to ameliorated cognitive deficits after cortical infarction.

Antiproliferative and proapoptotic effects of epigallocatechin gallate on human leiomyoma cells

DIAPHRAGM NEUROSTIMULATION RESULTS IN LOWER TRANSPULMONARY DRIVING PRESSURE AND HIPPOCAMPAL APOPTOSIS, WITH MODERATE CORRELATION, IN A MODERATE-ARDS PRECLINICAL MODEL